A robust approach to estimate relative phytoplankton cell abundances from metagenomes

Phytoplankton account for >45% of global primary production, and have an enormous impact on aquatic food webs and on the entire Earth System. Their members are found among prokaryotes (cyanobacteria) and multiple eukaryotic lineages containing chloroplasts. Genetic surveys of phytoplankton communities generally consist of PCR amplification of bacterial (16S), nuclear (18S) and/or chloroplastic (16S) rRNA marker genes from DNA extracted from environmental samples. However, our appreciation of phytoplankton abundance or biomass is limited by PCR-amplification biases, rRNA gene copy number variations across taxa, and the fact that rRNA genes do not provide insights into metabolic traits such as photosynthesis. Here, we targeted the photosynthetic gene psbO from metagenomes to circumvent these limitations: the method is PCR-free, and the gene is universally and exclusively present in photosynthetic prokaryotes and eukaryotes, mainly in one copy per genome. We applied and validated this new strategy with the size-fractionated marine samples collected by Tara Oceans, and showed improved correlations with flow cytometry and microscopy than when based on rRNA genes. Furthermore, we revealed unexpected features of the ecology of these ecosystems, such as the high abundance of picocyanobacterial aggregates and symbionts in the ocean, and the decrease in relative abundance of phototrophs towards the larger size classes of marine dinoflagellates. To facilitate the incorporation of psbO in molecular-based surveys, we compiled a curated database of >18,000 unique sequences. Overall, psbO appears to be a promising new gene marker for molecular-based evaluations of entire phytoplankton communities.     

Experimental tendon repair: glycosaminoglycan arrangement in newly synthesized collagen fibers.

Changes in the macromolecular orientation and metachromasy of glycosaminoglycans (GAG) in newly synthesized and assembled collagen fibers in rat Achilles tendon after tendon excision were investigated in toluidine blue (TB)-stained preparations, based in the selective absorption of polarized light (= linear dichroism, LD) and of absorption of unpolarized light in situ. Extrinsic LD was observed microspectrophotometrically from the early phases of tendon repair onwards, although the absorption peaks in both parallel and perpendicular directions with respect to the plane of polarized light and the long axis of the collagen fibers occurred at the same wavelength, and thus differed from the pattern situation in normal adult controls. Compared to normal adult tendons, the pattern of LD in newly synthesized and assembled fibers was still not fully attained 110 days after surgical tendon removal. This incomplete recovery possibly reflected the influence of aging during the repair process. There was no correlation between LD and metachromasy. The highest absorption values for metachromatic staining occurred on the 7th day after tendon removal, at a time when LD was not intense. Treatment with hyaluronidase showed that the LD in the early stages of tendon repair was mostly due to hyaluronate whereas the LD in the later stages was due to chondroitin sulfates. The changes in LD during Achilles tendon repair were attributed to gradual modifications in the composition and macromolecular orientation of GAGs relative to the long axis of the collagen fibers.     

Solid-phase Synthesis and Cellular Localization of a C- and/or N-terminal Labelled Peptide

We report the solid-phase synthesis by the Fmoc strategy of a peptide containing a cysteamide group at its C-terminus. This peptide was subject to further modifications including the linkage of fluorophores, namely lucifer yellow and coumarin respectively, at the C- and/or N-terminals. After incubation with living cultured cells these two probes were localized and it is concluded that the post-synthesis modifications can strongly modify the localization of the peptide.     

A Short-Term Decrease in Nitrogenase Activity (C2H2 Reduction) Is Induced by Exposure of Soybean Shoots to Their CO2 Compensation Point

Photosynthesis and nitrogenase acetylene-reducing activity (ARA) were measured in soybeans (Glycine max [L.] Merr.) in which the shoots were exposed for 48 h to 60 [mu]L L-1 CO2, a value corresponding to their CO2 compensation point. Six hours after the beginning of the light period at low CO2, the ARA started to decrease, reaching a rate of 50% of the control rate in 14 to 24 h and 20% of the control rate in 34 to 38 h after the beginning of the CO2 treatment. At these times, there was no net photosynthesis, and the transpiration rate was 20% lower than that in the control plants. An increase in the partial pressure of O2 around the nodules alleviated this inhibition of ARA. The maximal ARA achieved at 40 kPaO2 was 3 times higher than that at 20 kPa O2 and similar to the maximal ARA of the control plants. It was argued that the decrease in ARA of soybean exposed to the CO2 compensation point was due to a decrease in the nodule's permeability to O2 diffusion.     

Water flux through a semi-deciduous forest grove of the Orinoco savannas

Water relations were analysed in a semi-deciduous forest grove occurring in the oxisols of the Orinoco savannas. This grove has a shallow unconsolidated ironstone cuirass, which is overlaid by a sandy loam layer (0.0–0.5 m) that contains more than 90% of the grove forest root phytomass. Evapotranspiration and through drainage were calculated by using data from the soil profile as related to physical characteristics of the site root zone, hydraulic conductivity, volumetric water content and potential hydraulic gradient. Mean annual evapotranspiration was 783 mm year^–1 and annual through drainage below the root zone was 14% (162 mm year^–1) of the gross rainfall. This drainage recharged the 42% of the annual saturation deficit of the water table. Similar mean annual evapotranspiration (770 mm year^–1) was also calculated by using the water balance components. The mean daily coupling omega factor () between the grove canopy and the surrounding atmosphere indicated that a high degree of coupling (=0.14±0.16) occurs in the grove and evapotranspiration was mainly controlled by surface conductance. As the dry season proceeded, the soil saturation deficit () increased rapidly resulting in a threshold surface conductance (0.030–0.005 m s^–1) for ranging from 0.05 to 0.10. Hypotheses to explain the omnipresence of perennial species in the wide range of physical conditions in neotropical savannas are discussed.     

The mite Varroa jacobsoni does not transmit American foulbrood from infected to healthy colonies

The present study was conducted to determine whether Varroa jacobsoni can transmit American foulbrood (AFB), caused by the bacterium Paenibacillus larvae to healthy colonies by the surface transport of spores. Five two-storey Langstroth colonies of Apis mellifera ligustica were infested by placing a sealed brood comb, with 10% Varroa prevalence, between the central brood combs of each colony. Two months later the colonies were inoculated with P. larvae by adding brood comb pieces with clinical signs of AFB (45±5 scales per colony). After 60 days the brood area was completely uncapped by means of dissecting needles and tweezers, separating the Varroa mites from the larvae and the collected mites were introduced at a rate of 51 per colony into four recipient hives placed in an isolated apiary. Twenty female Varroa specimens were separated at random and observed by SEM. Paenibacillus larvae spores were found on the dorsal shield surface and on idiosomal setae. All colonies died after 4–5 months due to a high incidence of varroosis. No clinical AFB symptoms or P. larvae spores were observed in microscopic preparations. It is concluded that Varroa jacobsoni does not transmit AFB from infected to healthy colonies; it does, however transport P. larvae spores on its surface.     

An easy method for the removal of Epon resin from semi-thin sections. Application of the avidin-biotin technique

Summary A simple procedure is described for removing Epon resin from semi-thin 1 m sections, which permits excellent postembedding immunohistochemical staining (avidin-biotin complex technique). The procedure was developed for the detection of growth hormone and prolactin in bovine adenohypophysis fixed with 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 m sodium cacodylate buffer pH 7.4–7.6. The results indicate that the removal of the epoxy embedding medium prior to the application of the immunohistochemical reagents was essential for the successful localization of the antigenic determinants of the two hormones. The immunocytochemical reactivity was obtained only after treating the sections with a solution of potassium hydroxide in a mixture of absolute methyl alcohol and propylene oxide (Maxwell's solution). An enhanced immunoreactivity was obtained when this treatment was followed by an additional treatment with either 4% hydrogen peroxide or a saturated aqueous solution of sodium metaperiodate. Because of the easy preparation of the Epon removal solution and the good structural preservation without damage to the antigenic determinants, Maxwell's solution is suggested as a good etching agent which can be used in immunohistochemical studies on semi-thin sections with excellent results.     

Salicylic acid and the plant pathogen Erwinia carotovora induce defense genes via antagonistic pathways

Infection of tobacco plants with the plant pathogenic bacterium Erwinia carotovora subsp. carotovora or treatment of plants with Erwinia -derived elicitor preparations leads to the induction of a number of genes thought to play a role in plant defense response to pathogens. In order to determine the role of salicylic acid (SA) in the induction of the Erwinia responsive genes, the accumulation of mRNAs for these and other genes encoding pathogenesis-related proteins (PR genes) in response to both Erwinia elicitors and SA was determined. PR genes were identified which were preferentially induced by Erwinia elicitor preparations, one gene was induced by SA but not by Erwinia , and another gene was induced by both type of treatments. The differential expression of these genes and the timing of induction suggest that SA is not the signal molecule leading to the early response of plants to Erwinia . This was demonstrated by experiments using transgenic NahG plants that overproduce a salicylate hydroxylase inactivating SA. The elicitation of PR genes by Erwinia was similar in NahG and wild-type plants. Therefore, induction of plant defense genes by Erwinia and SA seems to be by two distinct pathways leading to expression of separate sets of genes. Furthermore, we could demonstrate that Erwinia elicitors antagonize the SA-mediated induction of PR genes. Similarly, SA appeared to inhibit the induction of PR genes elicited by Erwinia . The observed antagonism between the two signal transduction pathways indicates the presence of a common regulatory element in both pathways that acts downstream of SA in the SA-mediated response.     

Glycosylation of Acetylcholinesterase Forms in Microsomal Membranes from Normal and Dystrophic Lama2dy Mouse Muscle

Abstract: The distribution and glycosylation of acetylcholinesterase (AChE) forms in vesicles derived from sarcoplasmic reticulum of normal muscle (NMV) were investigated and compared with those from dystrophic muscle vesicles (DMV). AChE activity was similar in NMV and DMV. Most of the AChE in NMV and half in DMV were released with Triton X-100. Asymmetric (A₁₂) and globular hydrophilic and amphiphilic (G^H₄, G^A₄, G^A₂, and G^A₁) AChE species occurred in NMV and DMV, the lighter forms being predominant. The percentage of G^H₄ and G^A₄ decreased in DMV. A fraction of the AChE that could not be extracted with detergent was detached with collagenase. Most of the detergent-released A₁₂ AChE from NMV and nearly half in DMV failed to bind to Ricinus communis agglutinin (RCA-I). Conversely, the collagenase-detached isoforms bound to RCA, revealing that asymmetric AChE associated with internal membranes or basal lamina differed in glycosylation. Moreover, nearly half of G^A₄ AChE in DMV and a few in NMV bound to RCA. Most of the RCA-unreactive G^A₄ forms in NMV come from sarcolemma. The results indicate that dystrophy induces minor changes in the distribution and glycosylation of AChE forms in internal membranes of muscle.